In addition to mediating sexual maturation and reproduction through stimulation of classical intracellular receptors that bind DNA and regulate gene expression, estradiol is also thought to influence various brain functions by acting on receptors localized to the neuronal membrane surface. Many intracellular signaling pathways and modulatory proteins are affected by estradiol via this unconventional route, including regulation of the transcription factor cAMP response element-binding protein (CREB). However, the mechanisms by which estradiol acts at the membrane surface are poorly understood. Because both estradiol and CREB have been implicated in regulating learning and memory, we characterized the effects of estradiol on this transcription factor in cultured rat hippocampal neurons. Within minutes of administration, estradiol triggered mitogen-activated protein kinase (MAPK)-dependent CREB phosphorylation in unstimulated neurons. Furthermore, after brief depolarization, estradiol attenuated L-type calcium channel-mediated CREB phosphorylation. Thus, estradiol exhibited both positive and negative influences on CREB activity. These effects of estradiol were sex specific and traced to membrane-localized estrogen receptors that stimulated group I and II metabotropic glutamate receptor (mGluR) signaling. Activation of estrogen receptor ␣ (ER␣) led to mGluR1a signaling, triggering CREB phosphorylation through phospholipase C regulation of MAPK. In addition, estradiol stimulation of ER␣ or ER␤ triggered mGluR2/3 signaling, decreasing L-type calcium channel-mediated CREB phosphorylation. These results not only characterize estradiol regulation of CREB but also provide two putative signaling mechanisms that may account for many of the unexplained observations regarding the influence of estradiol on nervous system function.

SUMMARY Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events but how information is relayed onward to the nucleus remains unclear. Here we report a novel mechanism that mediates long-distance communication within cells: a shuttle that transports Ca2+/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, that its phosphorylation at Thr287 by βCaMKII protects the Ca2+/CaM signal, and that CaN triggers its nuclear translocation. Both βCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, while γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca2+/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves longstanding puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII and CaN in multiple neuropsychiatric disorders.

Activity-dependent gene expression triggered by Ca2+ entry into neurons is critical for learning and memory, but whether specific sources of Ca2+ act distinctly or merely supply Ca2+ to a common pool remains uncertain. Here we report that both signaling modes co-exist and pertain to CaV1 and CaV2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. CaV1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca2+ to drive local CaMKII aggregation and trigger communication with the nucleus. By contrast, CaV2 channels must elevate [Ca2+]i microns away and promote CaMKII aggregation at CaV1 channels. Consequently, CaV2 channels are ∼10-fold less effective in signaling to the nucleus than CaV1 channels for the same bulk [Ca2+]i increase. Furthermore, CaV2-mediated Ca2+ rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca2+, further attenuating CaV2-mediated gene expression.

Communication between cell surface proteins and the nucleus is integral to many cellular adaptations. In the case of ion channels in excitable cells, the dynamics of signaling to the nucleus are particularly important because the natural stimulus, surface membrane depolarization, is rapidly pulsatile. To better understand excitation–transcription coupling we characterized the dependence of cAMP response element–binding protein phosphorylation, a critical step in neuronal plasticity, on the level and duration of membrane depolarization. We find that signaling strength is steeply dependent on depolarization, with sensitivity far greater than hitherto recognized. In contrast, graded blockade of the Ca2+ channel pore has a remarkably mild effect, although some Ca2+ entry is absolutely required. Our data indicate that Ca2+/CaM-dependent protein kinase II acting near the channel couples local Ca2+ rises to signal transduction, encoding the frequency of Ca2+ channel openings rather than integrated Ca2+ flux—a form of digital logic.

A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.